The cow udder is divided into the left and the right by a thick medial suspensory ligament, and also into the front and the rear by thin membranes that operate as partition wall to constitute independent quarters. The cow udder contains a large number of mammary alveoli, and a single layer of mammary epithelial cells is arranged at the mammary gland side of each mammary alveolus. Mammary epithelial cells of a healthy cow are tightly connected by inter-cellular adhesion molecules such as tight junctions. Mammary epithelial cells form a layer, which functions as barrier for physically separating the inside and the outside of each of the mammary glands. The layer containing mammary epithelial cells prevents flowing substances from blood to milk or from milk to blood, and thus prevents the mutual influx of substances between milk and blood.
Mammary epithelial cells mainly bear (1) a function of synthesizing milk proteins and lactose and secreting them to the alveolar lumen of the mammary glands, (2) a function of transferring lipid droplets to the apical site of epithelial cells and projection of them on the cell surface to secret butterfat into the alveolar lumen and (3) a function of transferring serum albumin and immunoglobulin from blood into milk.
Generally, epithelial cells of skin and mucous membrane are involved in the defense mechanism of preventing the invasion of foreign objects, such as pathogenic microorganisms, to living bodies. Epithelial cells not only physically prevent invasions of pathogens into living bodies but also chemically inhibit invasions and proliferations of pathogenic microorganisms by lactic acid, mucin, lysozyme, antibacterial peptide and so on that epithelial cells produce. Epithelial cells are also involved in inducing early immune reactions by producing various cytokines, in addition to the above physical and chemical barriers.
On the fluctuations of appearance of leukocytes and mammary epithelial cells in milk, CD4+T cells that induce production of antibodies are mainly contained at dry periods, whereas cytotoxic γδ+T cells and CD8+T cells are mainly contained at lactation periods. In contrast, B cells and antibody producing cells that are involved in humoral immunity are practically contained in milk (see Non-Patent Documents 1 through 3 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). Macrophages (Mφ), dendritic cells (DC), granulocytes are involved in the elimination mechanism of infected epithelial cells. In addition to the phagocytic cells, it has been reported that γδ+T cells and CD8+T are involved actively in cell-mediated immunity at lactation periods (see Non-Patent Documents 4 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). Therefore, a considerable attention is attracted in a natural immunity for the immune system of mammary gland, unlike other organs where IgA is immediately produced by B cells, such as a intestinal mucosal immune system (see Non-Patent Documents 5 and 6 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention).
Mastitis is an inflammatory disease that is occurred after the invasion of pathogens into udders. After microorganisms such as bacteria, molds and enzymes invade an udder through the teat orifice of a mammary gland, the inflammation arises and then it develops mastitis in the mammary gland. An udder quarter with mastitis shows hypertrophy as a whole and the mammary tissue thereof is destroyed, if partly, when compared with a normal udder quarter. Symptoms of mastitis include (1) an increase in the number of somatic cells in the milk secreted from the infected udder quarter that arises as a result of mobilization of immune cells and (2) a fall in the quantity and quality of milk due to interstitial hypertrophy and atrophy of mammary alveoli. Individual cow suffering from mastitis can show general symptoms including appetite loss and diarrhea in addition to the above described symptoms and eventually die in some instances.
Mastitis is roughly divided into clinical mastitis and latent mastitis, which does not give rise to any clinical symptom but shows an increase in the number of somatic cells in milk. Latent mastitis is believed to be responsible for about 80% of the damage cost caused by mastitis. Escherichia coli, which is a Gram negative bacterium, causes serious inflammations which result in clinical mastitis, whereas Gram positive bacteria such as Staphylococcus aureus frequently cause latent mastitis. The frequency of clinical expressions of latent mastitis is relatively low, it is difficult to discover and medically treat latent mastitis. The problem is that the latent mastitis can spread among hosts of cows without being noticed. Additionally, latent mastitis can be aggravated to turn into clinical mastitis in some instances.
A major therapeutic method of mastitis is administration of antibiotic agent. However, when it comes to mastitis caused by Staphylococcus aureus, part of Staphylococcus aureus is resistant against antibiotic substances and can form minute abscesses in mammary glands to make it generally difficult to medically treat mastitis caused by Staphylococcus aureus by means of antibiotic agents.
Antibiotic agents are therapeutically effective in terms of prevention of infections. Since, however, administration of antibiotic agents is a symptomatic therapy, it cannot directly protect mammary gland tissues from damages. Other known therapeutic methods for mastitis include those employing physiologically active substances such as cytokines (GM-CSF, CXCL8, hIFN-α) and those employing natural substances showing anti-bacterial effects (Stevia extract fermentation products, defensins, BIMURON®), although some of them are still in experimental stages.
The PL test that employs changes in pH and in the viscosity in milk as indicators, which occur as a result of coagulation and denaturation due to the number of leucocytes in milk, is commonly utilized as a method of diagnosing mastitis. Additionally, there is the somatic cell count test method of using as indicator the number of immune cells released into milk due to the pathogenic bacteria that have invaded a mammary gland. Furthermore, the method of detecting as light the weak electric potential that appears when somatic cells are activated and using it as indicator, namely the chemical luminescence (CL) measurement method is also known (see Non-Patent Documents 14 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). The CL measurement method is based on the principle of measuring the active oxygen release quantity of neutrophils by way of chemical luminescence. With regard to mastitis, some cases on fluctuations of lymphocyte subsets in milk have been reported, which are caused by the fact that immune cells such as lymphocytes that have reacted to bacteria in milk infiltrate in milk (see Non-Patent Documents 8 and 9 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention).
In immune responses to inflammations, migration and supply of leukocytes take an important role in the immunological monitoring of hosts in inflammations. Leukocyte migration is a phenomenon where leukocytes that have been stimulated by chemotactic factors such as chemokines, cytokines and physiologically active lipids, which are secreted during inflammation, infiltrate into tissues from blood vessels, move and accumulate to sites of inflammation. Various regulatory factors for leukocyte migrations are known. Among them, chemokines that belong to the chemotaxis factor cytokine family are mainly known. Other known factors include cyclophilins, which are one of chemotaxis factors. Cyclophilins appear in all cells of all living things including procaryotes and eucaryotes. Cyclophilins are generally intracellular proteins and show peptidyl-prolyl cis-trans-isomerase activity, which is a factor that is involved in folding of proteins. Cyclophilins are known as FK-506 binding proteins. Two of cyclophilins including 18 kDa cyclophilin A (CyPA) and 21 kDa cyclophilin B (CyPB) are dominant. Cyclophilin A is a cytoplasmic protein having no signal sequence, while cyclophilin B is linked to endoplasmic reticulum in the N-terminal signal sequence (see Non-Patent Documents 10 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention).
CyPA is a cyclophilin that exists most massively, and is believed to account for about 0.1 to 0.4% by quantity of the total intracellular proteins. CyPA is known as a member of the protein group belonging to the intracellular binding factors of cyclosporine A, which is an immunosuppressive agent (see Non-Patent Documents 11 and 12 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). During the onset of inflammation, CyPA is also released and secreted from both dead cells and living cells and exerts its function outside cells (see Non-Patent Documents 13 through 16 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). Particularly, CyPA that is secreted to the outside of cells causes migrations of leukocyte subsets such as monocytes, eosinophil granulocytes, neutrophils and T-lymphocytes (see Non-Patent Documents 13, 17 and 18 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). Rises of CyPA levels in diseases such as sepsis, rheumatism, arthritis, pneumonia and aneurism have been reported (see Non-Patent Documents 19 and 20 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). With regard to rheumatism, there are reports telling that the CyPA level and the number of neutrophils are correlated (see Non-Patent Documents 21 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). Similarly, CyPA appears and are secreted in pulmonary epithelial cells and in vascular endothelial cells in some diseases including pneumonia and arterial aneurysm. Reports telling that extracellular CyPa is a powerful chemotaxis factor to monocytes, neutrophils, eocinophil granulocytes and T-lymphocytes of human are known (see Non-Patent Documents 21 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention). There are also known reports telling that CyPA can induce inflammations because it is released due to an acute inflammatory reaction such as mobilization of neutrophils when bacteria invade into living bodies (see Non-Patent Documents 11 listed below, the contents of which are incorporated herein by reference for the purpose of supporting the disclosure of the present invention).